Advantageous salts of mu-opiate receptor peptides

ABSTRACT

The subject invention provides advantageous new salts of mu-opiate receptor peptides. These salts have been found to have excellent properties in terms of their crystal structure, stability, solubility, lack of impurities and/or the ability to be produced, with these advantageous properties, in amounts sufficient for the production of therapeutic compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The subject application is a continuation of co-pending application Ser.No. 16/929,994, filed Jul. 15, 2020; which is a continuation ofapplication Ser. No. 16/016,165, filed Jun. 22, 2018; which is acontinuation of application Ser. No. 14/570,432, filed Dec. 15, 2014;which is a continuation of application Ser. No. 14/056,496, filed Oct.17, 2013; now U.S. Pat. No. 8,940,704; which is a continuation ofapplication Ser. No. 12/744,859, filed on Jul. 22, 2010; which is aNational Stage Application of International Application No.PCT/US2008/086838, filed on Dec. 15, 2008; which claims the benefit ofU.S. Provisional Application Ser. No. 61/007,617, filed Dec. 13, 2007,all of which are incorporated herein by reference in their entirety.

The Sequence Listing for this application is labeled“September2010-ST25.txt”, which was created on Sep. 30, 2010, and is 9KB. The entire content is incorporated herein by reference in itsentirety.

BACKGROUND OF INVENTION Field of the Invention

This invention relates to salts of peptides that bind with high affinityand selectivity to the mu (morphine) opiate receptor; pharmaceuticalpreparations containing an effective amount of the peptide salts; andmethods for providing analgesia, relief from gastrointestinal disorderssuch as diarrhea, and therapy for drug dependence containing aneffective amount of the peptide salts.

Description of the Related Art

Many peptides have been found that exhibit opiate-like activity bybinding to opiate receptors. Three different types of opiate receptorshave been found: delta (δ), kappa (κ) and mu (μ). The major putativefunction for opiates is their role in alleviating pain. Other areaswhere opiates are well-suited for use in treatment are conditionsrelating to gastrointestinal disorders, schizophrenia, obesity, bloodpressure, convulsions, and seizures. Although the δ and κ receptors mayalso mediate analgesia, activation of μ receptors is the primary andmost effective means of inducing analgesia, and is the primary mechanismby which morphine acts.

Because morphine and other compounds with clinical usefulness actprimarily at the μ receptor, pharmaceutical compositions having peptideswith high affinity and selectivity for this site are of considerableimportance. It would be desirable to produce these peptide compositionsin a simple, efficient, and economical manner.

BRIEF SUMMARY

The subject invention provides advantageous new salts of mu-opiatereceptor peptides. These salts have been found to have excellentproperties in terms of their crystal structure, stability, solubility,lack of impurities and/or the ability to be produced, with theseadvantageous properties, in amounts sufficient for the production oftherapeutic compositions.

Specifically exemplified herein are aspartate, maleate, lactate andhydrochloride salts of mu-opiate receptor peptides. In a particularlypreferred embodiment, the subject invention provides hydrochloride saltsof endomorphin peptides.

The peptides that can be used according to the subject invention havethe general formula Tyr-X₁-X₂-X₃ wherein X₁ is Pro, D-Lys or D-Orn; X₂is Trp, Phe or N-alkyl-Phe wherein alkyl contains 1 to about 6 carbonatoms; and X₃ is Phe, Phe-NH₂, D-Phe, D-Phe-NH₂ or p-Y-Phe wherein Y isNO₂, F, Cl or Br.

In a specific advantageous embodiment, the subject invention providesthe hydrochloride salt of a cyclic endomorphin-1 peptide (designatedherein as CYT-1010).

The subject invention further provides pharmaceutical compositionscomprising these advantageous salts.

The subject invention further provides therapeutic methods that utilizethe salts and compositions described herein.

The subject invention further provides methods for preparing the saltsof the subject invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows x-ray diffractogram of CYT-1010 free base lot V05060N1.

FIG. 2 shows DSC/TGA overlay of CYT-1010 free base lot V05060N1.

FIG. 3 shows H-NMR spectra of CYT-1010 free base lot V05060N1.

FIG. 4 shows FTIR spectrum of CYT-1010 free base lost V05060N1.

FIGS. 5A-5B show DVS moisture isotherm of CYT-1010 free base.

FIG. 6 shows x-ray diffractogram of the primary screen aspartate salt.

FIG. 7 shows DSC/TGA overlay of the primary screen aspartate salt.

FIG. 8 shows x-ray diffractogram of the primary screen hydrochloridesalt.

FIG. 9 shows DSC/TGA overlay of the primary screen hydrochloride salt.

FIG. 10 shows x-ray diffractogram of the primary screen lactate salt.

FIG. 11 shows DSC/TGA overlay of the primary screen lactate salt.

FIG. 12 shows x-ray diffractogram of the primary screen maleate salt.

FIG. 13 shows DSC/TGA overlay of the primary screen maleate salt.

FIG. 14 shows x-ray diffractograms of the aspartate salt: primary screensample (blue trace), scaled-up sample (black trace).

FIG. 15 shows DSC/TGA thermograms of the scaled-up sample aspartatesalt.

FIG. 16 shows DSC overlay of the primary screen sample (upper trace) andthe scaled-up sample (lower trace) of the aspartate salt.

FIG. 17 shows H-NMR spectra of the scaled-up sample aspartate salt.

FIG. 18 shows FTIR spectrum of the scaled-up aspartate.

FIGS. 19A-19B show DVS moisture isotherm of the scaled-up aspartate.

FIG. 20 shows x-ray diffractograms of the hydrochloride: primary screensample (black trace), scaled-up sample (red trace).

FIG. 21 shows DSC/TGA overlay of the scaled-up hydrochloride salt.

FIG. 22 shows DSC overlay of the primary screen sample (lower trace) andthe scaled-up sample (upper trace) of the hydrochloride salt.

FIG. 23 shows H-NMR spectra of the scaled-up sample hydrochloride.

FIG. 24 shows FTIR spectrum of the scaled-up hydrochloride.

FIGS. 25A-25B show DVS moisture isotherm of the scaled-up hydrochloridesalt.

FIG. 26 shows TGA thermograms comparison of CYT-1010 HCl before andafter 25° C./75% RH exposure.

FIG. 27 shows x-ray diffractograms of CYT-1010 HCl (black) and waterslurry sample (blue trace).

FIG. 28 shows x-ray diffractograms of the lactate: primary screen sample(black trace), scaled-up sample (red trace).

FIG. 29 shows DSC/TGA overlay of the lactate scaled-up sample.

FIG. 30 shows DSC overlay of the primary screen sample (upper trace) andthe scaled-up sample (lower trace) of the lactate salt.

FIG. 31 shows H-NMR spectra of the scaled-up lactate.

FIG. 32 shows FTIR spectrum the scaled-up lactate.

FIGS. 33A-33B show DVS moisture isotherm of the scaled-up lactate salt.

FIG. 34 shows x-ray diffractograms of the maleate: primary screen sample(black trace), scaled-up sample (red trace)

FIG. 35 shows DSC/TGA overlay of the maleate scaled-up sample.

FIG. 36 shows DSC overlay of the primary screen sample (upper trace) andthe scaled-up sample (lower trace) of the maleate salt.

FIG. 37 shows H-NMR spectrum of the scaled-up sample of the maleatesalt.

FIG. 38 shows spectrum of the scaled-up sample of the maleate salt.

FIGS. 39A-39B show DVS moisture isotherm of the scaled-up maleate salt.

FIGS. 40A-40D show particle morphology of four scaled-up salts.

FIG. 41 shows XRD data for HCl salt.

FIG. 42 shows DSC/TGA data for HCl salt.

FIG. 43 shows DSC comparison of the scaled-up sample (upper trace) and asmall scale sample (lower trace).

FIG. 44 shows XRD data for aspartate salt.

FIG. 45 shows DSC/TGA data for aspartate salt.

FIG. 46 shows DSC data. Scaled-up aspartate (upper trace), small scalesample (lower trace.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO:1 is a peptide useful according to the subject invention.

SEQ ID NO:2 is a peptide useful according to the subject invention.

SEQ ID NO:3 is a peptide useful according to the subject invention.

SEQ ID NO:4 is a peptide useful according to the subject invention.

SEQ ID NO:5 is a peptide useful according to the subject invention.

SEQ ID NO:6 is a peptide useful according to the subject invention.

SEQ ID NO:7 is a peptide useful according to the subject invention.

SEQ ID NO:8 is a peptide useful according to the subject invention.

SEQ ID NO:9 is a peptide useful according to the subject invention.

SEQ ID NO:10 is a peptide useful according to the subject invention.

SEQ ID NO:11 is a peptide useful according to the subject invention.

SEQ ID NO:12 is a peptide useful according to the subject invention.

SEQ ID NOS:13-26 are additional peptides useful according to the subjectinvention.

DETAILED DISCLOSURE

The subject invention provides advantageous salts of peptides that bindto the mu (morphine) opiate receptor with high affinity, selectivity andpotency.

Advantageously, the salts of the subject invention have excellentproperties in terms of their crystallinity, morphology, thermalproperties, stoichiometry, hydroscopicity, aqueous solubility and/orchemical stability.

This invention also provides pharmaceutical preparations containing aneffective amount of one or more of the peptide salts. The subjectinvention further provides methods for providing analgesia, relief fromgastrointestinal disorders such as diarrhea, anti-inflammatorytreatments, and therapy for drug dependence wherein the methods involveadministering, to a patient in need of such treatment, a compositioncontaining an effective amount of one or more of the peptide salts ofthe subject invention.

Initially, 16 salts of a cyclic endomorphin-1 peptide analog wereselected for evaluation. These included fifteen monosalts and onehemisalt. Characterization of these salts on a 50 mg scale allowed theidentification of four particularly advantageous salts: the aspartate,hydrochloride, maleate, and lactate salts.

The hydrochloride salt exhibited good crystallinity. The stoichiometryof the hydrochloride salt based on ion chromatography was close totheoretical. The monosalt appears to form a stable monohydrate at above5% RH. Chemical stability appears excellent as well. Accordingly, in aparticularly preferred embodiment, the subject invention provides thehydrochloride salt of endomorphin-1 (and analogs thereof) as well aspharmaceutical compositions that contain this salt.

Peptides

The peptides that can be used according to the subject invention havethe general formula Tyr-X₁-X₂-X₃ wherein X₁ is Pro, D-Lys or D-Orn; X₂is Trp, Phe or N-alkyl-Phe wherein alkyl contains 1 to about 6 carbonatoms; and X₃ is Phe, Phe-NH₂, D-Phe, D-Phe-NH₂ or p-Y-Phe wherein Y isNO₂, F, Cl or Br. Some preferred peptides of the invention are:

(SEQ ID NO: 1) H-Tyr-Pro-Trp-Phe-NH₂ (SEQ ID NO: 2)H-Tyr-Pro-Phe-Phe-NH₂ (SEQ ID NO: 3) H-Tyr-Pro-Trp-Phe-OH (SEQ ID NO: 4)H-Tyr-Pro-Phe-Phe-OH (SEQ ID NO: 5) H-Tyr-Pro-Trp-D-Phe-NH₂(SEQ ID NO: 6) H-Tyr-Pro-Phe-D-Phe-NH₂ (SEQ ID NO: 7)H-Tyr-Pro-Trp-pNO₂-Phe-NH₂ (SEQ ID NO: 8) H-Tyr-Pro-Phe-pNO₂-Phe-NH₂(SEQ ID NO: 9) H-Tyr-Pro-N-Me-Phe-Phe-NH₂ (SEQ ID NO: 10)H-Tyr-Pro-N-Et-Phe-Phe-NH₂ (SEQ ID NO: 11) H-Tyr-Pro-N-Me-Phe-D-Phe-NH₂(SEQ ID NO: 12) H-Tyr-Pro-N-Et-Phe-D-Phe-NH₂ (SEQ ID NO: 13)H-Tyr-c-[D-Lys-Trp-Phe] (SEQ ID NO: 14) H-Tyr-c-[D-Lys-Phe-Phe](SEQ ID NO: 15) H-Tyr-c-[D-Orn-Trp-Phe] (SEQ ID NO: 16)H-Tyr-c-[D-Orn-Phe-Phe] (SEQ ID NO: 17) H-Tyr-c-[D-Lys-Trp-pNO₂-Phe](SEQ ID NO: 18) H-Tyr-c-[D-Lys-Phe-pNO₂-Phe] (SEQ ID NO: 19)H-Tyr-c-[D-Orn-Trp-pNO₂-Phe] (SEQ ID NO: 20)H-Tyr-c-[D-Orn-Phe-pNO₂-Phe] (SEQ ID NO: 21)H-Tyr-c-[D-Lys-N-Me-Phe-Phe] (SEQ ID NO: 22)H-Tyr-c-[D-Orn-N-Me-Phe-Phe] (SEQ ID NO: 23)H-Tyr-c-[D-Lys-N-Et-Phe-Phe] (SEQ ID NO: 24)H-Tyr-c-[D-Orn-N-Et-Phe-Phe] (SEQ ID NO: 25)H-Tyr-c-[D-Lys-N-Me-Phe-D-Phe] (SEQ ID NO: 26)H-Tyr-c-[D-Lys-N-Et-Phe-D-Phe]

The last fourteen peptides listed are cyclic peptides whose linearprimary amino acid sequences are given in SEQ ID NO:13 through SEQ IDNO:26. In this context, the applicants incorporate herein by reference,in its entirety, U.S. Pat. No. 6,303,578.

The peptide of SEQ ID NO:1 is highly selective and very potent for the.mu.opiate receptor, with over 4000-fold weaker binding to deltareceptors and over 15,000-fold weaker binding to kappa receptors,reducing the chances of side-effects.

The peptides of this invention may be prepared by conventionalsolution-phase (Bodansky, M., Peptide Chemistry: A Practical Textbook,2^(nd) Edition, Springer-Verlag, New York (1993) or solid phase(Stewart, J. M.; Young, J. D. Solid Phase Peptide Synthesis, 2^(nd)edition, Pierce Chemical Company, 1984) methods with the use of properprotecting groups and coupling agents. A suitable deprotection methodmay then be employed to remove specified or all of the protectinggroups, including splitting off the resin if solid phase synthesis isapplied.

Cyclization of the linear peptides can be performed by, for example,substitution of an appropriate diamino carboxylic acid for Pro inposition 2 in the peptides through ring closure of the 2-position sidechain amino and the C-terminal carboxylic functional groups. Thecyclization reactions can be performed with the diphenylphosphoryl azidemethod (Schmidt, R., Neuhert, K., Int. J Pept. Protein Res. 37:502-507,1991).

Peptides synthesized with solid phase synthesis can be split off theresin with liquid hydrogen fluoride (HF) in the presence of the properantioxidant and scavenger.

The amount of the reactants utilized in the reactions, as well as theconditions required to facilitate the reactions and encourage efficientcompletion may vary widely depending on variations in reactionconditions and the nature of the reactants.

The desired products may be isolated from the reaction mixture bycrystallization, electrophoresis, extraction, chromatography, or othermeans. However, a preferred method of isolation is HPLC. All of thecrude peptides can be purified with preparative HPLC, and the purity ofthe peptides may be checked with analytical HPLC. Purities greater than95% of the synthesized compounds using HPLC have been obtained.

In a preferred embodiment specifically exemplified herein, the peptideis that which is shown as SEQ ID NO:13 (cyclic endomorphin-1 peptide)and has the following structure:

Pharmaceutical Compositions

The present invention also provides pharmaceutical preparations thatcontain a pharmaceutically effective amount of the peptide salts of thisinvention and a pharmaceutically acceptable carrier or adjuvant. Thecarrier may be an organic or inorganic carrier that is suitable forexternal, enteral or parenteral applications.

The peptide salts of the present invention may be compounded, forexample, with the usual non-toxic, pharmaceutically acceptable carriersfor tablets, pellets, capsules, liposomes, suppositories, intranasalsprays, solutions, emulsions, suspensions, aerosols, targeted chemicaldelivery systems (Prokai-Tatrai, K.; Prokai, L; Bodor, N., J. Med. Chem.39:4775-4782, 1991), and any other form suitable for use. The carrierswhich can be used are water, glucose, lactose, gum acacia, gelatin,mannitol, starch paste, magnesium trisilicate, tale, corn starch,keratin, colloidal silica, potato starch, urea and other carrierssuitable for use in manufacturing preparations, in solid, semisolid,liquid or aerosol form, and in addition auxiliary, stabilizing,thickening and coloring agents and perfumes may be used.

Therapeutic Methods

The present invention also provides methods for providing analgesia,relief from gastrointestinal disorders such as diarrhea, and therapy fordrug dependence in patients, such as mammals, including humans, whichcomprises administering to the patient an effective amount of thepeptides, or salts thereof, of this invention. The diarrhea may becaused by a number of sources, such as infectious disease, cholera, oran effect or side-effect of various drugs or therapies, including thoseused for cancer therapy. For applying the peptide salts of the presentinvention to human, it is preferable to administer them by parenteral orenteral administration.

The peptide salts of the subject invention can also be used to provideanti-inflammatory treatments. In this context the applicants incorporateherein by reference, in its entirety, U.S. 2004/0266805.

The dosage of effective amount of the peptides varies from and alsodepends upon the age and condition of each individual patient to betreated. However, suitable unit dosages may be between about 0.01 toabout 100 mg. For example, a unit dose may be from between about 0.2 mgto about 50 mg. Such a unit dose may be administered more than once aday, e.g. two or three times a day.

Experimental Methods

Morphology. A Zeiss Universal microscope configured with a polarizedvisible light source was used to evaluate the optical properties of thesamples. Specimens were typically mounted on a microscope slide.Magnification was typically 125×. Observations of particle/crystal sizeand shape were recorded. The presence of birefringence was also noted.

Stoichiometry—¹H-NMR. Samples were prepared by dissolving 3-7 mg indimethylsulfoxide (DMSO)-d₆ with 0.05% (v/v) tetramethylsilane (TMS).Spectra were collected at ambient temperature on a Varian Gemini 300 MHzFT-NMR spectrometer.

Stoichiometry—Ion Chromatography. Standards solutions were preparedgenerally in the 5 to 50 μg/mL range. The samples were dissolved andanalyzed with a Dionex DX-600 ion chromatograph configured with aAminiPac PA 10 column for asparatic acid analysis and AS4A-SC/AG4A-SCcolumn for maleic, hydrochloric and lactic acids and a conductivitydetector.

Solubility. The solubility of the selected primary screen salts wasdetermined at ambient temperature in aqueous buffer pH 7 by a visualtechnique. The solubility of the scaled-up salts was visually determinedin aqueous pH 4.7 and 10 buffers both by the visual technique and HPLCanalysis alongside with the stability samples using the samechromatographic condition (see section HPLC analysis).

Thermal Properties by Differential Scanning Calorimetry (DSC). DSC datawere collected on a TA Instruments 2910 DSC. In general, samples in themass range of 1 to 10 mg were crimped in aluminum sample pans andscanned from 25° C. to past the melt at 10° C./minute using a nitrogenpurse at 50 mL/min.

Thermal Properties by Thermogravimetric Analysis (TGA). TGA data werecollected on a TA Instruments 2950 TGA. In general, samples in the massrange of 5 to 15 mg were placed in an open, pre-tared platinum samplepan and scanned from 25 to about 300° C. at 10° C./minute using anitrogen purge.

Optical by Hot Stage Microscopy (HSM). A Zeiss Universal microscopeconfigured with a polarized visible light source and a Mettler hot stageaccessory was used to analyze CYT-1010 free base. A sample was mountedon a microscope slide with a drop of immersion oil and a cover glass.Magnification was 400×. Sample was heated from 25° C. to about 300° C.at 3 or 10° C./minute. Observations of phase change, recrystallization,evolution of bubbles, etc. were recorded.

Crystallinity by X-Ray Powder Diffraction (XRD). Diffraction patternswere collected using a Bruker D8 Discovery diffractometer configuredwith an XYZ stage, laser videomicroscope for positioning, and HiStararea detector. Collection times were 120 seconds at room temperature. ACu Kα radiation 1.5406 Å tube was operated at 40 kV and 40 mA. The X-rayoptics consist of a Gobel mirror coupled with a pinhole collimator of0.5 mm. Theta-theta continuous scans were employed with asample-detector distance ofd 15 cm, which gives an effective 20 range of4-40°. Samples were mounted in low background quartz plates (9 mmdiameter, 0.2 mm deep cavity).

Infrared Spectroscopy (FTIR). Infrared spectra were obtained with aNicolet 510 M-O Fourier transform infrared spectrometer, equipped with aHarrick Splitpera™ attenuated total reflectance device. Spectra wereacquired from 4000-400 cm⁻¹ with a resolution of 4 cm⁻¹, and 128 scanswere collected for each analysis.

Solution Stability. The four final salt candidates were dissolved (induplicate) in acetonitrile:water: (90:10) with 0.1% TFA at aconcentration of 0.3 mg (free base basis) per mL of solvent. Thescintillation vials were sealed. One vial of each salt solution wasplaced in a 40™ C over for 2 weeks. Another vial of each salt solutionwas stored at 25° C. for 2 week. These solutions (and “time zero”solutions) were analyzed for CYT-1010 by HPLC.

Solid State Stability. Power samples of the four final salt candidateswere transferred (in duplicate) to scintillation vials and sealed. Onevial of each salt was placed in a 60° C. over for 2 week. Another vialof each salt was stored at 25° C. for 2 week. These samples (and “timezero” solutions) were analyzed for CYT-1010 by HPLC.

Photo Stability. Samples of the four final salt candidates weretransferred (in duplicate) to crystallizing dishes and sealed withSaran® wrap. One dish of each salt was also covered with aluminum foil(as the dark controls). Another dish of each salt was not covered withaluminum foil (as the photoexposed samples). The samples were exposed toICH compliant option 2 UV sources to examine their stability withrespect to light at approximately 25° C. Dark controls and time zerosamples were also analyzed for comparison. Samples were analyzed forCYT-1010 by HPLC.

Oxidation Stability. Samples of the four final salt candidates wereexposed to a pure oxygen atmosphere for 2 weeks to examiner theirstability with respect to oxidation at 25° C. Samples were analyzed forCYT-1010 by total area normalization for impurity profile by HPLC.

HPLC Analysis. Salt candidates were analyzed by total area normalization(TAN). The samples were dissolved in acetonitrile:water (90:10) with0.1% TFA at a free base concentration of 0.3 mg/mL.

HPLC Conditions

HPLC column: YMC-Pack ODS-A 150 mm, 4.6 mm, 5 micron

Guard Column (optional) None

Column Temp: 25±1° C.

Sample Temp: ambient

Autosampler Flush: 1:1 Water:CAN

Flow Rate: 1.5 mL/min

Injection Volume: 7 μL

UV Detection: 215 nm

Run Time: 32 minutes

Analysis Time: 32 minutes

Mobil Phase: A—0.1% TFA in water

-   -   B—0.1% TFA in CAN    -   Gradient Puym Program*:

Step Time Elapsed Time % A % B (minutes) (minutes) (Aqueous) (organic)Curve 0.5 0.0 90 10 0 15.0 15.5 5 95 1 5.0 20.5 5 95 0 6.0 26.5 90 10 16.0 32.5 90 10 0

Dynamic Vapor Sorption (DVS) (Performed by Surface Measurement SystemsLtd., Allentown, Pa.). Samples were run in an automated dynamic vaporsorption analyzer from 0 to 95% relative humidity and back to 0%relative humidity at 25° C. in 5% RH steps. Samples were predried (toconstant mass) under a dry nitrogen stream before analysis. Weightchange as a function of humidity and time was recorded to construct amoisture isotherm and kinetic plot of water sorption and desorption.Sample masses were generally in the range of 1-5 mg.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1—Initial Evaluation

Fifteen acids were selected for detailed evaluation. The acids usedduring the study are shown in Table 1.

TABLE 1 Acetic Aspartic (L) Citric Fumaric Gluconic (D) HippuricHydrochloric Lactic Malic Maleic Mucic Phosphoric Sulfuric SuccinicTartaric (L)

Salts were initially prepared on an approximately 50 mg scale. The freebase was suspended in methanol. All acids, except aspartic and mucicwere dissolved in water. Equal molar portions of the free base and acidsolutions were mixed to form the monosalts. Molar portions of the freebase and half-molar acid solutions were mixed to form the hemisalt.Aspartic and mucic acid were added as dry powders as they werewater-insoluble. Free base suspensions after additions of hydrochloric,sulfuric, maleic, phosphoric, tartaric, and citric acids became clearand were then evaporated while stirring on a stirplate at ambienttemperature. The remaining cloudy salt preparations were left stirringcapped for approximately two days to allow the reactions to occur, thenevaporated the same way while stirring on a stirplate at ambienttemperature. Salts were vacuum dried at 40° C.

Fifteen salts of the free base were prepared and evaluated for thermalproperties and crystallinity. The different salt forms are shown inTable 2.

TABLE 2 Salt Forms Evaluated Acetate Hippurate Mucate Tartrate (L)Aspartate (L) Hydrochloride Phosphate Gluconate (D) Citrate LactateSulfate Maleate Fumarate Malate (L) Hemi-sulfate Succinate

Example 2—Characterization of Free Base

XRD analysis indicated that the free base was crystalline as shown inFIG. 1. An overlay of DSC and TGA thermograms can be seen in FIG. 2. TheDSC thermogram exhibited multiple thermal events: a small endotherm withan onset temperature of 256.2° C. and an enthalpy value of ΔH=20 J/gimmediately followed by a small exotherm. The second endothermic peakwas a sharp peak with an onset temperature of 286.3° C. and an enthalpyvalue of 95.7 J/g. TGA thermogram exhibited the weight loss due tovolatiles of 2.9 wt % (25° C.-150° C.).

Hot stage microscopy analysis indicated that the particles of the freebase were irregularly shaped, platy and did not appear birefringent. Themelting of the sample was completed by approximately 288° C. No otherthermal events were evident.

H-MHR and FTIR spectra of the free base are shown in FIG. 3 and FIG. 4,respectively.

DVS analysis of the free base indicated non-stoichiometric water uptakeof up to 1.7 wt % by 90% RH (FIG. 5).

The stability data of the free base did not exhibit significant changesin HPLC-TAN values upon exposure to heat, light, or oxygen (Tables 6-9).

Example 3—Characterization of Primary Salts

Fifteen monosalts and one hemisalt were prepared and analyzed by powderX-ray diffraction. The thermal behavior of the primary salts was studiedusing DSC and TGA analysis. The analytical results are summarized inTable 3. As can be seen from the table, some salts were crystalline,others were low-ordered or amorphous. The crystallinity of some saltswas improved by slurrying them in water for approximately a week. Thethermal behavior of some salts was complex with the multiple thermalevents observed on heating. Endotherms due to dehydration and/ordesolvation for some of the salts were also evident.

TABLE 3 Crystallinity and Thermal Data of Selected Salts TGA resultsSalt Crystallinity by XRD DSC results (weight loss) HCI MonoLow-ordered, was ripened by Small endotherm at Weight loss 3% bystirring in water, crystallintiy 230° C. with ΔH = 150° C. improved. 10J/g, melt with decomposition at 283° C. Sulfate Hemi Amorphous, wasripened by stirring in water, remained amorphous. Sulfate Monocrystalline Broad endotherm at ~100° C., Weight loss 5% by melt withdecomposition 150° C. and at 283° C. continued until melt Aspartate (L)Mono Crystalline Endotherm onset: 270° C., 1.4% (25-150° C.). ΔH = 241J/g Stable weight up (melt + decomposition). to 225° C. Maleate MonoLow-ordered, was ripened by Endotherm onset: Weight loss 2.3% stirringin water, crystallinity 237° C., ΔH = 95 J/g by 150° C., and improved.dramatic wt. loss after 150° C. Phosphate Mono Crystalline Broadendotherm at ~200° Weight loss 2.2% C. with ΔH = 40 by 150° C., and J/g,melt with dramatic wt. loss decomposition at 308° after 175° C. Tartrate(L) Mono Crystalline Broad endotherm at 2.0% (25-150° C.) 86° C., smallendotherm 139° C., with with decomposition at 252° C. Fumarate MonoCrystalline Endotherm onset: 263° 5.3% (25-175° C.) C., ΔH = 111 J/g,Mucate Mono Crystalline A broad endotherm 2.7% (25-150° C.) at ~75-100°C., a double endotherm at 207° C. Citrate Mono Amorphous, was ripened bystirring in water, remained amorphous. Malate (L) Mono CrystallineEndotherm onset: 260.3° 1.0% (25-150° C.), C., ΔH = 157 J/g, losingweight loss after 150° C. and up to the melt Hippurate Mono Low-ordered,was ripened by stirring in water, crystallinity improved, but XRDpattern matched free base starting material, salt did not form.Gluconate (D) Mono XRD pattern matched free base starting material, saltdid not form. Lactate (L) Mono Crystalline Endotherm onset: 234° 3.0%(25-150° C.) C., ΔH = 116 J/g, Succinate Mono Low-ordered, was ripenedby A broad endotherm 2.5% (25-150° C.) stirring in water, crystallinityat ~75° C., a double improved. endotherm at 248° C. Acetate Mono Twosamples generated: XRD of one sample matched free base, other sample waslow- ordered.

Solubility. The solubility of selected salts from the primary screen wasdetermined. Solubility measurements were made at ambient temperature inan aqueous pH 7.0 buffer. The results of the solubility measurements areshown in Table 4. Even at a concentration of 0.05 mg/ml, the solutionswere still cloudy for all salts indicating the solubility of all saltswas <0.05 mg/mL. Solutions of the lactate, malate and aspartate saltsappeared less hazy/cloudy than others, suggesting their solubility maybe slightly higher than the other forms.

TABLE 4 Visual Solubility of Primary Salts in Aqueous pH 7.0 Buffer SaltVisual Solubility (mg/ml) Aspartate (L) <0.05 Tartrate (L) <0.05 Maleate<0.05 Malate <0.05 Phosphate <0.05 Lactate (L) <0.05 Succinate <0.05Hydrochloride <0.05

The evaluation of the primary salts are summarized in Table 5. Thisevaluation allowed the selection of four salts for further evaluation:aspartate, hydrochloride, lactate and maleate.

TABLE 5 Summary of Salt Forms of the Primary Salt Screen Salt FormComments Aspartate (L) Single melting endotherm, small amount ofvolatiles, appears slightly more soluble than most other salts forms.Maleate Slightly more soluble than most other salt forms, TGA indicatedthermal instability after 150° C. Lactate (L) Was not as soluble as someothers, sample had 3% volatiles, single melting endotherm. HydrochlorideCrystallinity was improved by ripening, 3% volatiles, multiple events inDSC. Tartrate (L) XRD pattern was very similar to maleate, was slightlymore soluble, but small additional endotherm in DSC trace could indicatepolymorphic behavior. Malate XRD pattern was very similar to tartrate,was slightly more soluble, single endotherm, small amount of volatiles,but loss of weight after 150° C. Succinate Salt did not crystallizewell, had to be ripened by slurring, one of the less soluble salts,sample had 2.5% volatiles, a double endotherm in DSC. Sulfate Sample had5% volatiles. Fumarate TGA weight loss 5.3%. Mucate Not a promising saltbased on thermal behavior. Phosphate One of the less soluble, multipleendotherms, weight loss profile is not good.Salt Screen Scale-Up. Based on the results of the primary salt screen,four monosalts were selected for a scale-up to the 400 mg scale:aspartate, hydrochloride, lactate and maleate. The same preparativeprocedure was used for the scale-up as was for the primary saltevaluation.

The characteristics of the four most promising salt forms are describedbelow.

Aspartate

The material was a white crystalline solid. The X-ray diffractionpattern of the batch is shown in FIG. 6. The DSC thermogram exhibited amelting endotherm with an extrapolated onset temperature ofapproximately 270° C. It appears to melt with decomposition. The totalvolatiles by TGA in a temperature range 25-150° C. were 1.4 wt %. ADSC/TGA overlay plot is shown in FIG. 7.

Hydrochloride

The hydrochloride salt isolated was a white crystalline solid. The X-raydiffraction pattern of the batch is shown in FIG. 8. The materialexhibited a small (8 J/g) endotherm with an onset temperature of 230° C.and the main endotherm with an onset of 282.7° C. (the DSC/TGA overlayis shown in FIG. 9). The weight loss observed using TGA at 150° C. was2.9 wt %.

Lactate

The XRD pattern of the lactate salt is shown in FIG. 10, it was lesscrystalline than the aspartate or hydrochloride. It was a white solid.The DSC thermogram revealed a single melting endotherm with an onsettemperature of 234° C. and an enthalpy value of 116 J/g. The sample lostapproximately 2.9 wt % at 150° C. by TGA as shown in FIG. 11.

Maleate

The malate was a slightly off-white crystalline solid, the XRD patternis shown in FIG. 12. The DSC thermogram had an endotherm with an onsettemperature of 236.7° C. and a heat of fusion 93.9 J/g. The TGAthermogram indicated a weight loss of 150° C. of 2.3 wt % but the samplestarted to loose mass at approximately 150° C. The DSC/TGA thermogramoverlay plot is shown in FIG. 13.

Example 4—Characterization of Scaled-Up Salts

After the initial evaluation, additional amounts of four salts(aspartate, hydrochloride, lactate, and maleate) were prepared forfurther evaluation. These salts were scaled up to approximately 400 mgto facilitate additional testing and determine if the characteristics ofthe solids were reproducible. The results of the scaled-up analyses aresummarized below.

Aspartate. The material produced in the scale-up batch was analyzed byXRD, DSC, TGA, H-NMR, and FTIR.

XRD overlay of the scaled-up aspartate salt (black trace) with a smallscale batch (blue) is shown in FIG. 14. The DSC/TGA data for thescaled-up aspartate is shown in FIG. 15. FIG. 16 shows the DSC overlayplot of the small scale sample with the scaled-up sample. The scaled-upsample appears to be less crystalline than the small scale sampledespite the fact that the additional ripening in water was done toimprove crystallinity. The DSC thermogram shows a single melt with anearlier melting onset than the small scale sample, provably due to thelower crystallinity. The sample had 1.7 wt % volatiles.

The aspartic acid content determined by ion chromatography wasapproximately 13.5 wt %, somewhat lower than the theoretical value formonosalt (17.6 wt %). This may have contributed to the lowercrystallinity of the sample.

The proton FT-NRM spectrum of the aspartate was collected and is shownin FIG. 17. The aspirate aliphatic peaks were over lapped by theCYT-1010 aliphatic peaks, so that molar ratio could not be determined byNMR.

The FTIR spectrum of the scaled-up aspartate is shown in FIG. 18.

DVS analysis of the scaled-up aspartate salt was performed. Theaspartate salt may form a hemihydrate then monohydrate at higherhumidity. The moisture sorption isotherm and kinetic data plot are shownin FIG. 19. The shape of the isotherm plot makes it difficult to becertain whether hydrates form.

The aqueous solubility results at pH 4, pH 7, pH 10 are shown in Table10.

Hydrochloride

The scaled-up sample of the hydrochloride salt displayed the same XRDpattern as in the initial evaluation (see XRD plots overlay for the twosamples in FIG. 20). The thermal behavior of both batches was alsosimilar. The DSC/TGA thermograms of the scaled-up sample is in FIG. 21and a comparison of DSC thermograms of the primary and scaled-up samplesis in FIG. 22. The material exhibited a small (10.2 J/g) endotherm withan onset temperature of 231.3° C. and the main endotherm with an onsetof 285.9° C. The sample had lost 2.6 wt % volatiles at 150° C.

Hot stage microscopy of the hydrochloride revealed no changes inparticle morphology up to the melt, which was observed at approximately280° C. The evolution of bubbles was evident at 108° C. and again at230° C.

The promoton NMR spectrum (FIG. 23) suggests that the stoichiometry ofthe salt is approximately 1:1. The FTIR spectrum of the scaled-uphydrochloride salt is shown in FIG. 24.

IC was used to evaluate the chloride content of the scaled-up salt. TheIC indicated the chloride content was 5.2 wt % (theoretical 5.5 wt %).

The DVS analysis suggested a reversible hydrate formation at 5% RH (FIG.25). Given the approximately 2.5 wt % water uptake, this would imply amonohydrate forms (theoretical 2.7 wt % water).

A sample of HCl was stored at 25° C. and 75% RH for one week and thenreweighed, no weight gain was observed. The exposed sample was analyzedby TGA to check the thermal profile after the exposure of the sample tohumidity. It did not show any additional weight loss due to moisture. Acomparison of TGA thermograms before and after the humidity exposure isshown in FIG. 26.

A slurry of the hydrochloride salt in water was carried out by stirringan excess of the HCl salt in water on a stirplate for approximately oneweek. Slurry was filtered and the wet cake analyzed by XRD to check forany structural changes. XRD patterns of samples before and after theslurry were essentially the same (FIG. 27), suggesting that the XRDpattern probably represents the monohydrate form of the material.

The stability data of the hydrochloride salt is described below.

The aqueous solubility results at pH 4, pH 7, and pH 10 are shown inTable 10.

Lactate

The scaled-up sample of the lactate salt was crystalline and had thesame XRD pattern as in the initial evaluation. Thermal profiles werealso similar (see XRD plots overlay for the two samples in FIG. 28 andDSC/TGA data for the scaled-up sample in FIG. 29). DSC thermogram of thescaled-up sample had a melting endotherm with an onset temperature ofapproximately 237.5° C. and an enthalpy value of 143 J/g. The totalvolatiles by TGA were 1.7 wt % at 150° C. A comparison of DSCthermograms of the primary and scaled-up samples is shown in FIG. 30.

The stoichiometry of the monosalt was evaluated using ion chromatography(IC). The sample contained approximately 9.8 wt % lactic acid which islower than the theoretical expected result (12.6 wt %). To determine whyit was lower, a lactic acid solution used for the preparation of salts(Fisher, assay 88.3%) was analyzed by IC and assay value was determinedto be only 76.8 wt % as opposed to the label claim of 88.3 wt %.

MNR and FTIR spectra of the scaled-up lactate are in FIG. 31, and FIG.32, respectively. The methine peak at ˜4.0 ppm integrated to 1.23. Thiswould indicate that the mole ratio is approximately 1 to 1 (a monosalt).

DVS analysis indicated non-stoichiometric water update of 1-3 wt % in0-70% RH range and up to 6 wt % by 90% RH (FIG. 33). Given the smoothshape of the curve it was not possible to deduce whether this uptakerepresents hydrate formation or not.

The aqueous solubility results at pH 4, pH 7, and pH 10 are shown inTable 10.

Maleate

The scaled-up sample of the maleate salt was crystalline. XRD patternsof the primary screen and scale-up malate salts were very similar as canbe seen in FIG. 34.

The DSC thermogram exhibited the same thermal behavior as in the initialevaluation. The weight loss observed at 125° C. was ˜2 wt %. Afteradditional drying, the volatile content was 1.5 wt %. The DSC/TGAoverlay plot of the scaled-up maleate is shown in FIG. 35. A comparisonof DSC thermograms of the primary and scaled-up samples is shown in FIG.36. The thermograms show good repeatability.

The stoichiometry of the monosalt was evaluated using ion chromatography(IC). The sample contained approximately 14.0 wt % maleic acid which isclose to the theoretical value of 15.7 wt %.

The H-NMR spectrum of the maleate is shown in FIG. 37. The FTIR spectrumof the scaled-up maleate salt is in FIG. 38.

DVS analysis (FIG. 39) indicated non-stoichiometric water uptake ofapproximately 3.5 wt % over the 0-95% RH range. Whether a hydrate formedor not could not really be deduced from this data.

The aqueous solubility results at pH 4, pH 7, and pH 10 are shown inTable 10.

Example 5—Stability of Scaled-Up Salts

The scaled-up aspartate, hydrochloride, lactate, and maleate saltstogether with the free base were analyzed in duplicate by total areanormalization (TAN) to determine their impurity profiles. The salts werestressed in solid state using heat, light, and a pure oxygen atmosphereto determine if the salt forms exhibited different chemical stabilitycharacteristics. The salts were also stressed in solution using heat.

Samples were prepared at a free base concentration of 0.3 mg/mL. Thediluent for all sample preparations was 90:10 acetronitrile:water with0.1% TFA. All solutions were sonicated for at least five minutes priorto analysis. Analysis was done over four days. The impurity profile ofthe free base, as shown in the table below, was consistent over thistime.

CYT-1010 Free Base Impurity Profile¹ Form 0.89 0.92 0.95 1.02 1.03 1.141.18 Free Base Day 1 0.09 0.05 ND 0.53 0.16 ND ND Free Base Day 2 0.120.06 ND 0.51 0.13 ND ND Free Base Day 3 0.13 0.07 ND 0.53 0.18 ND ND¹Impurity profile an average of the first three injections of the WIFree Base Injections.

Solution Stability

The solution stability characteristics were evaluated by collecting HPLCdata on solutions stored in sealed vials for two weeks at approximately25 and 40° C. The storage solution consisted of 90:10acetronitrile:water with 0.1% TFA which is the diluent for the HPLCassay. Table 6 summarizes the results of the HPLC analyses of theseexperiments.

TABLE 6 Solution Stability Results Time Zero 2 weeks at 25° C. 2 weeksat 40° C. Salt (area %) (area %) (area %) Free Base 99.2 98.6 98.7L-aspartate 99.1 98.6 98.6 Maleate 96.2 96.7 97.2 Lactate 99.2 98.5 98.5Hydrochloride 99.1 98.5 98.5

The solution stability data for the free base and three of four salts,L-aspartate, lactate, and hydrochloride, showed a decrease between 0.6and 0.7 area % at 25° C. These compounds exhibited almost the samedecrease in area percent at 40° C. This behavior suggests that thechanges for these compounds were due to exposure to the diluent and notthe heat. On the other hand, the maleate salt showed a 0.5 area %increase at 25° C. and an additional 0.5 area % increase at 40° C.

Solid State Stability

The solid state stability characteristics were evaluated by collectingHPLC data on salt samples stored in sealed vials at 25 and 60° C. fortwo weeks. Results of the HPLC analyses are summarized in the Table 7.

TABLE 7 Solid State Stability Results Time Zero 2 weeks at 25° C. 2weeks at 60° C. Salt (area %) (area %) (area %) Free Base 99.2 98.8 98.7L-aspartate 99.1 99.1 98.9 Maleate 96.2 96.2 89.1 Lactate 99.2 99.0 98.5Hydrochloride 99.1 98.9 98.9

The thermal stability data for the L-asparate and maleate salts did notexhibit significant changes in assay values upon exposure at 25° C. Thelactate and hydrochloride salts only decreased slightly, a decrease of0.2 area %, while the free base decreased 0.4 area % at 25° C. Thehydrochloride salt did not exhibit further decrease in area % at 60° C.The free base decreased slightly in area % and the lactate saltdecreased 0.5 area % at 60° C. as compared to 25° C. data. The maleatesalt showed significant decrease, 7.1 area %.

Photostability

Samples of the four salt candidates were exposed to ICH compliant option2 UV sources to examine their stability with respect to light atapproximately 25° C. Dark controls were also analyzed for comparison.Table 8 summarizes the results of the HPLC data on the photostabilitysamples

TABLE 8 Photostability Stress Results 2 weeks Dark 2 weeks UV Time ZeroControl Exposed Salt (area %) (area %) (area %) Free Base 99.2 99.0 98.9L-aspartate 99.1 99.1 98.8 Maleate 96.2 96.3 96.3 Lactate 99.2 99.0 98.6Hydrochloride 99.1 99.1 99.0

The photostability data for the two of the four salts did not exhibitsignificant changes in assay values upon exposure. The lactate saltshowed the greatest change in area %, a decrease of 0.6 area % from timezero to exposed, while the free base and L-asparate salt both decreased0.3 area % from time zero to exposed.

Oxidation Stability

Samples of the four salts were exposed to a pure oxygen atmosphere toexamine their stability with respect to oxidation at 25° C. Table 9summarizes the results of the HPLC data on the photostability.

TABLE 9 Oxidation Stability Results Salt Time Zero (area %) 2 weeksOxidation (area %) Free Base 99.2 97.7 L-aspartate 99.1 98.8 Maleate96.2 95.8 Lactate 99.2 98.8 Hydrochloride 99.1 98.7

The oxidative stability data for the free base had the greatest changewith 1.5 area % decrease. The four salts showed a decrease between 0.3and 0.4 area %.

Example 6—Solubility of Scaled-Up Salts

Solubility measurements were made at ambient temperature in pH 4 buffer(potassium bisphthalate buffer 0.05 molar), pH 7 buffer (potassiumphosphate mono basic-sodium hydroxide buffer 0.05 molar) and pH 10buffer (potassium carbonate-potassium hydroxide buffer 0.05 molar). Twoapproaches were tried. A visual technique and HPLC analysis were used todetermine the solubilities.

By visual technique, solubilities of all four salts and the free base inpH 4, 7 and 10 buffers were less than 0.05 mg/ml.

The results of the solubility determinations by HPLC are shown in Table10. HPLC data were collected on solutions stored in sealed vials forapproximately one week at 25° C. at pH 4, pH 7, and pH 10. Portions ofthese solutions were filtered with a Teflon 0.45 micro filter prior toHPLC analysis. The results were calibrated with a six point calibrationcurve ranging from 0.12 to 0.003 mg/ml. The same HPLC conditions wereused as listed previously except the injection volume was increased to10 μL.

TABLE 10 Solubility of Salts in Aqueous Buffers pH Results - mg/ml SaltpH 4 pH 7 pH 10 Free Base ND <0.003 0.006 L-aspartate ND <0.003 0.007Maleate ND <0.003 0.007 Lactate ND <0.003 0.007 Hydrochloride ND <0.0030.006

The solubility behavior was similar for all five compounds in that thesolubility increased with increasing pH.

Example 7—HPLC Impurity Profiles

Time Zero. Some observations were made concerning the HPLC impurityprofile of the free base and salt samples. First, impurities at 0.89,0.92, 1.02, and 1.03 RRT (relative retention time) are detected in thetime zero sample preparations of the free base and all four salts. Thelargest of these four impurities is the 1.02 RRT impurity which isbetween 0.4 and 0.5 area % in all five compounds. The maleate salt alsohas a very large impurity peak at time zero (2.7 area %) at 1.14 RRT.Thus the maleate salt has a relatively low area % purity of 96 area %.Time zero impurity profile data is shown in Table 11.

TABLE 11 Impurity Profile at Time Zero Impurity by RRT Salt 0.87 0.890.92 0.95 1.02 1.03 1.14 1.18 Free Base ND 0.09 0.05 ND 0.53 0.16 ND NDL-aspartate ND 0.05 0.09 0.07 0.46 0.10 0.12 <0.03 Maleate ND 0.11 0.130.16 0.40 0.18 2.67  0.11 Lactate ND 0.04 0.08 0.10 0.49 0.13 ND <0.03Hydrochloride <0.03 0.04 0.08 0.19 0.49 0.09 ND ND

Example 8—Stability Solution Stability

The solution stability data for the free base and three out of foursalts, L-asparate, lactate, and hydrochloride, showed a decrease between0.6 and 0.7 area % at 25° C. and 60° C. In all the stability solutions,including the maleate, a new impurity peak appears at 0.21 RRT that is0.5 to 0.6 area % in size. This peak is unique to the solution stabilitysamples. Another difference is seen with the maleate salt. The peak at1.14 RRT decreases from a time zero value of 2.7 area % to 1.7 area % at25° C. in solution and to 1.1 area % at 40° C. in solution. This is thesame impurity peak that increases to 10 area % in the 60° C. solid statemaleate sample. The solution stability impurity profile data is shown inTables 12 and 13.

TABLE 12 Solution Stability 25° C. Impurity Profile Impurity by RRT Salt0.21 0.89 0.92 0.95 0.98 1.02 1.03 1.14 1.18 Free Base 0.50 0.06 0.040.06 ND 0.53 0.18 ND ND L-aspartate 0.52 ND 0.08 0.12 ND 0.48 0.16 0.07ND Maleate 0.62 0.04 0.09 0.18 ND 0.42 0.16 1.71 0.07 Lactate 0.59 0.050.11 0.14 ND 0.49 0.15 ND ND Hydrochloride 0.59 ND 0.04 0.21 0.50 0.14ND ND

TABLE 13 Solution Stability 40° C. Impurity Profile Impurity by RRT Salt0.21 0.89 0.92 0.95 0.98 1.02 1.03 1.14 1.18 1.32 2.39 Free Base 0.500.04 ND 0.03 ND 0.51 0.18 ND ND ND ND L-aspartate 0.58 <0.03 0.07 0.13<0.03 0.47 0.15 ND ND ND ND Maleate 0.57 0.06 0.07 0.27 <0.03 0.43 0.151.14 0.05 ND 0.03 Lactate 0.62 0.04 0.04 0.14 <0.03 0.50 0.16 ND ND<0.03 <0.03 Hydrochloride 0.57 ND ND 0.23 0.03 0.51 0.15 ND ND ND 0.03

Solid State Stability

For the solid state stability samples, the free base at 25° C. and themaleate at 60° C. showed the greatest change in the impurity profile asshown in the tables below. For the free base at 25° C., the 0.89 and0.92 RRT impurities showed the largest increase as compared to time zerodata. For the maleate salt at 60° C., the 1.14 RRT peak increase from2.7 to 10 area % and while the impurity at 1.02 RRT decreased from 0.5area % to a nondetectable level.

Photostability

For the photostability samples, the lactate salt showed the greatestchange in the impurity profile. In the dark control, the 0.89 and 0.92RRT impurities increased as compared to time zero data while in thephotoexposed, the 0.92 and 0.95 RRT impurities increased significantly.

Oxidative Stability

For the oxidation samples, the free base showed the greatest change inthe impurity profile as shown in Table 14. The 0.89 and 0.92 RRTimpurities showed the largest increase as compared to time zero data.These two impurities increased for all of the salts as well.

TABLE 14 Oxiation Impurity Profile Impurity by RRT Salt 0.87 0.89 0.920.95 0.98 1.02 1.03 1.14 1.18 Free Base¹ 0.04 0.30 0.20 0.04 ND 0.530.20 ND ND L-aspartate ND 0.12 0.14 0.09 ND 0.48 0.16 0.09 <0.03 Maleate0.04 0.36 0.27 0.21 ND 0.38 0.15 2.70  0.12 Lactate <0.03  0.24 0.200.13 ND 0.49 0.15 ND <0.03 Hydrochloride 0.05 0.18 0.16 0.22 ND 0.520.18 ND ND ¹Different impurity profiles for the two sample preparations.

Morphology of Scaled-up Salts

The particle morphology of the four scaled-up salts was evaluated.Aspartate, hydrochloride and maleate particles were irregularly shaped,platy and did not appear birefringent. The lactate particles appearedlarger and not as thin as other salts (see FIG. 40).

Example 9—Properties of Scaled-Up Salts

Scale-up (on a 300 mg scale) of four salts was done: aspartate, maleatelactate, and hydrochloride. HCl and aspartate salts were analyzed byXRD, DSC, TGA.

XRD overlay of the scaled-up salt (red) with a small scale batch (black)is in FIG. 41. DSC/TGA data for the scaled-up HCl is in FIG. 42, FIG. 43shows the DSC overlay with a small scale sample. Both XRD and DSC datawere good matches to the small scale sample. The salt was relativelydry: amount of volatiles detected for HCl was 2.6%.

XRD overlay of the scaled-up salt (black) with a small scale batch(blue) is in FIG. 44. DSC/TGA data for the scaled-up aspartate is inFIG. 45, FIG. 46 shows the DSC overlay with a small scale sample.Scaled-up sample appears to be less crystalline than the small scalesample despite the fact that the additional ripening in water was doneto improve crystallinity. DSC shows a single melt with an earliermelting onset than the small scale sample, probably due to reduction incrystallinity. The sample had 1.7 wt % volatiles.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

What is claimed:
 1. A peptide salt wherein the peptide has a sequenceselected from SEQ ID NOs: 1-26 and the salt is selected from the groupconsisting of maleate, hydrochloride, and lactate.
 2. The peptide salt,according to claim 1, wherein the peptide is SEQ ID NO:13.
 3. Thepeptide salt, according to claim 2, wherein the salt is thehydrochloride salt.
 4. A pharmaceutical composition comprising a peptidesalt wherein the peptide has a sequence selected from SEQ ID NOs: 1-16and the salt is selected from the group consisting of maleate,hydrochloride, and lactate.
 5. The pharmaceutical composition, accordingto claim 4, wherein the peptide is SEQ ID NO:13.
 6. The pharmaceuticalcomposition, according to claim 5, wherein the salt is the hydrochloridesalt.
 7. The pharmaceutical composition, according to claim 4, furthercomprising 2-Hydroxypropyl-β-cyclodextrin.
 8. A method for treating acondition that is modulated by μ-opiate receptor activity, wherein saidmethod comprises administering, to a patient in need of such treatment apeptide salt wherein the peptide has a sequence selected from SEQ IDNOs: 1-26 and the salt is selected from the group consisting of maleate,hydrochloride, and lactate.
 9. The method, according to claim 8, whereinthe peptide is SEQ ID NO:13.
 10. The method, according to claim 9,wherein the salt is the hydrochloride salt.
 11. The method, according toclaim 8, which is used to provide analgesia or to treat a conditionselected from the group consisting of gastrointestinal disorders,inflammation, and drug dependence.
 12. The method, according to claim 8,wherein the composition further comprise 2-Hydroxypropyl-β-cyclodextrin.